The objective of Project 2 is to define the role of the lipin family of proteins in triglyceride metabolism. We previously identified mutations in lipin-1 as the cause of lipodystrophy in the fatty liver dystrophy mutant mouse strain, and established that lipin-1 is a determinant of adipose tissue development, obesity, and insulin sensitivity in mice and humans. Very recently, two distinct molecular functions for lipin-1 have been characterized. First, lipin-1 is a phosphatidate phosphatase-1 (PAP1) enzyme, catalyzing a key step in triglyceride biosynthesis, and accounting for all PAP1 activity in adipose tissue and skeletal muscle. Second, lipin-1 is a transcriptional coactivator of PPAR (peroxisome proliferator-activated receptor)-alpha and PPARgamma in hepatocytes and adipocytes. We have also identified two additional lipin family members and determined that they have PAP1 activity and exhibit prominent expression in liver (lipin-2) or bone (lipin-3). We hypothesize that each of the three lipin proteins has unique, tissue-specific roles in triglyceride metabolism through their actions as PAP1 enzymes and transcriptional coactivators. The Specific Aims are: (1) To determine the mechanisms by which point mutations in mouse lipin-1 and human lipin-2 impair PAP1 and/or coactivator function to cause lipodystrophy and the inflammatory disorder, Majeed syndrome, respectively. (2) To determine the requirement for lipin-1 coactivator versus PAP1 function in adipocyte differentiation, physiology, and metabolism using genetic and chemical manipulation of lipin-1 activity. We will generate cultured cells and mice that express "coactivator only" lipin-1 protein and determine ability of the mutant to complement lipin-1 deficiency in adipose tissue, liver, muscle, nerve, and reproductive function. We will also characterize the mechanism by which a novel adipogenic compound identified in Project 3 enhances PAP1 activity, and determine effects of other proadipogenic compounds on lipin-1 coactivator and PAP1 activity. (3) To establish the physiological roles of lipin-2 and lipin-3. We will generate knockout mouse models and characterize the role of lipin-2 in hepatic lipid metabolism and inflammation, and the role of lipin-3 in bone lipid metabolism and production of bone hormones that modulate systemic glucose homeostasis. The elucidation of lipin protein functions may lead to novel approaches for modulating adiposity, insulin sensitivity and inflammation contributing to obesity, hyperlipidemia, and diabetes.